A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys(2)-Cys(2) zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

醇溶蛋白盒（prolamin box，P-box）是一段高度保守的7碱基对序列元件（5′-TGTAAAG-3′），广泛分布于诸多谷类种子贮藏蛋白基因的启动子区域。玉米胚乳来源的核因子可与玉米醇溶蛋白基因（zeins）中的P-box发生特异性结合。P-box存在于所有玉米醇溶蛋白基因的启动子中，这提示胚乳DNA结合蛋白与P-box的相互作用，或许在玉米醇溶蛋白基因于胚乳发育阶段的协同激活表达过程中发挥关键调控作用。我们已克隆得到一个玉米胚乳特异性的互补脱氧核糖核酸（complementary DNA，cDNA），命名为醇溶蛋白盒结合因子（prolamin-box binding factor，PBF），其编码产物属于新近被报道的植物Cys₂-Cys₂锌指DNA结合蛋白Dof家族。凝胶迁移实验检测结果表明，PBF与P-box的序列特异性结合活性，与玉米胚乳核提取物中的内源因子完全一致。此外，PBF可在体外与碱性亮氨酸拉链蛋白Opaque2发生相互作用；Opaque2是一类已知的玉米醇溶蛋白基因表达转录激活因子，其结合靶点位于22 kDa玉米醇溶蛋白基因启动子的P-box下游20碱基对位置。PBF基因的成功分离与克隆，为进一步探究高度保守的P-box在调控谷类贮藏蛋白基因表达中的功能角色，提供了不可或缺的研究工具。